DNA probes for campylobacter, arcobacter and helicobacter

ABSTRACT

Nucleic acid probes and a method for their use in detecting and identifying Campylobacter, Helicobacter, and Arcobacter spp. bacterial pathogens are described. A method for prepg. specific nucleic acid probes for the detection of identification of any other bacterial pathogens is also provided.

This application claims benefit of Provisional Application 60/043,415filed Apr. 8, 1997.

FIELD OF THE INVENTION

The invention relates to nucleotide probes that are useful in detectingand identifying bacterial pathogens. More particularly, the inventionrelates to nucleotide probes that are useful in detecting andidentifying Campylobacter, Helicobacter and Arcobacter spp. bacterialpathogens.

BACKGROUND OF THE INVENTION

Campylobacter, Helicobacter, and Arcobacter spp. are examples of commonhuman and animal pathogens (Thomas, C. A. et al., 1966). Although thepathogenicity of such bacteria has long been known, their phylogeneticrelationships, isolation, detection, identification, and classificationby traditional biochemical tests, have been variable and difficult. Thisis largely due to their fastidious growth requirements, inability toferment carbohydrates, and diverse growth characteristics which vary,not only between genera and species, but also within species. Thus,their large phenotypic variations have made biochemical tests unreliableas a sole method for identifying and differentiating these bacteria.

Many of the species in the genera Helicobacter and Arcobacter were onceclassified under the genus Campylobacter. However, the phylogeneticrelationships of these bacteria have been reevaluated based oninformation from DNA-DNA hybridization, 23S rRNA-DNA hybridization(Vandamme et al., 1991; Vandamme et al., 1993), and partial 16S rRNAsequences (Li et al., 1993; Patton et al., 1991; Totten et al., 1987).These phylogenetic studies have led to the formation of the currentclassification of the Campylobacter and Vibrio organisms intoCampylobacter, Helicobacter, and Arcobacter.

Other than the conventional biochemical tests, alternative methods basedon molecular and genetic approaches, have been proposed to improve theidentification and differentiation of these bacteria to the specieslevel. These methods include serology (Hebert et al., 1983; Penner, J.L., 1988), enzymology (Elharrif, Z. and Megraud, F., 1986; Paster etal., 1991), cellular fatty acid compositions (Goodwin et al., 1985),electrophoretic protein patterns (Costas et al., 1987; Penner, J. L.,1988), random PCR-DNA fingerprinting (Eyers et al., 1993; Giesendorf etal., 1993; Giesendorf et al., 1994; and Vandamme et al., 1992), andDNA-DNA hybridization (Macario, A. J. L. and Macario, E. C. de. (eds.),1990; and Penner, J. L., 1988). A highly specific DNA-DNA hybridizationmethod is oligo hybridization. By varying hybridization conditions suchas ionic concentration and temperature, oligo probes can detect singlenucleotide sequence differences (Lee/Lane, 1992).

SUMMARY OF THE INVENTION

The present inventors have developed a method for preparing nucleic acidprobes for identifying species of bacterial pathogens, and havedeceloped nucleic acid probes for identifying species of Campylobacter,Helicobacter and Arcobacter.

In particular, the inventors have identified several probes that arespecific for the Campylobacter species, including Campylobacter jejuni(C.jejuni), Campylobacter coli (C.coli), Campylobacter lari (C.lari) andCampylobacter upsaliens (C.upsaliens); the Helicobacter species,including Helicobacter cinaedi (H. cinaedi); Helicobacter pylori(H.pylori); Helicobacter canis (H. canis); and the Arcobacter species,including Arcobacter nitrofigalis (A.nitrofigalis); Arcobacter butzleri(A. butzleri) and Arcobacter butzleri-like (A. butzleri-like).

The probes are useful in detecting the presence of a bacterial pathogenand are further useful in determining the identity of the specificpathogen.

In one aspect, the present invention relates to an isolated nucleic acidprobe for detecting or identifying C.jejuni. In one embodiment the probeis designated CJATC-1 and has the sequence 5′-TTTTC CGCAC ACTCA TGTAGTAAGC TCAAC TA-3′, and is identified as SEQ ID NO: 1. In anotherembodiment the probe is designated CJATC-2 and has the sequence 5′-GAAAAAGTAA GAGAA ATTGC TAAAA AAGAA-3′, and is identified as SEQ ID NO: 2.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying C. coli. In one embodiment theprobe is designated CC-1 and has the sequence 5′-ATTTC CTCAT GCTCA TGTAGTAAGC TCTAC AA-3′, and is identified as SEQ ID NO: 3. In anotherembodiment the probe is designated CC-2 and has the sequence 5′-GAAAAAGTTA GGGAA ATTGC TCATA TTGTA-3′, and is identified as SEQ ID NO: 4.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying C. lari. In one embodiment theprobe is designated CL-1 and has the sequence 5′-ATTCC CTTAT GCTCA TGTTGTAAGT TCT-3′, and is identified as SEQ ID NO:5. In another embodimentthe probe is designated CL-2 and has the sequence 5′-GATAA AGTTA GAGAGATAGC AAAAG AGATT-3′, and is identified as SEQ ID NO: 6.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying C. upsaliens. In one embodimentthe probe is designated CU-1 and has the sequence 5′-TTTCC CTCAC GCACACATCG TAAGC TCA-3′, and is identified as SEQ ID NO: 7. In anotherembodiment the probe is designated CU-2 and has the sequence 5′-GAAAAAGTAA GAGAA ATAGC ACACA TCGTT-3′, and is identified as SEQ ID NO: 8. Ina further embodiment the probe is designated GlyA-CU and has thesequence 5′-GGT TAG TAG CTC GGG TAA AAT GTA TGA AAG C-3′ and isidentified as SEQ ID NO: 15.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying H. cinaedi. In one embodimentthe probe is designated HC-1 and has the sequence 5′-TGAGC GCGTG AAGCAGCTAT TTGGC TGTGC GT-3′, and is identified as SEQ ID NO:9.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying H.pylori. In one embodiment theprobe is designated HP-1 and has the sequence 5′-AGAAA GGGCT AAAAA GCTTTTCAAT TGCCA GT-3′, and is identified as SEQ ID NO: 10.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying H. canis. In one embodiment theprobe is designated GlyA-HC and has the sequence 5′-CAG GAT TGA TTA CGACAA GCT ACG CCA AAG CGC GC-3′ and is identified as SEQ ID NO: 16. Inanother embodiment the probe is designated GlyA-HC2 and has the sequence5′-TTC TGC CTA TAC AAG AGA GCT AGA TTT TGC CAA G-3′ and is identified asSEQ ID NO: 17.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying A. nitrofigalis. In oneembodiment the probe is designated AN-1 and has the sequence 5′-AGATAGAGCT TGTGA AATTT TTGGT TGTAA AT-3′, and is identified as SEQ ID NO: 11.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying A. Butzleri. In one embodimentthe probe is designated GlyA-AB and has the sequence 5′-GCT TCT GCA TACGCA AGA GAA ATT GAT TCA AA- 3′ and is identified as SEQ ID NO: 12.

In another aspect, the present invention relates to an isolated nucleicacid probe for detecting or identifying A. Butzleri-like. In oneembodiment the probe is designated GlyA-BL and has the sequence 5′-GCAAGT GCA TAT GCA AGA GAG ATT GAT TTT AA-3′ and is identified as SEQ IDNO: 13. In another embodiment the probe is designated GlyA-BL2 and hasthe sequence 5′-AAG TAA ACC AAG CTT TTC AGG GCA AAA CTA CTC T-3′ and isidentified as SEQ ID NO: 14.

The nucleic acid probes of the present invention permit the detectionand identification of pathogenic bacteria in various samples includingbiological, food, or environmental samples.

Accordingly, the invention provides a method for detecting the presenceof a specific bacteria in a sample comprising contacting the nucleicacid molecules of the sample with a nucleic acid probe according to thepresent invention and determining if the sample hybridizes with thenucleic acid probe.

The invention further provides a kit for detecting the presence of aspecific bacteria in a sample comprising one or more nucleic acid probesaccording to the present invention, reagents required for hybridizationof the nucleic acid probe with the nucleic acid molecules in the sample,and directions for its use.

Other features and advantages of the present invention will becomeapparent from the following detailed description.

It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is a multiple nucleotide sequence alignment of the partial glyAsequences.

FIG. 2 shows PCR products of all species resolved in a 1% agarose geland used in the Southern hybridization experiments.

FIG. 3 shows autoradiographs of the Southern hybridizations testing thespecies-specificity of the series 1 probes.

FIG. 4 shows autoradiographs of the Southern hybridizations testing thespecies-specificity of the series 2 probes.

FIG. 5 shows sensitivity of the PCR/hybridization strategy.

FIG. 6 is a multiple nucleotide sequence alignment of the partial glyAsequences of Example 2.

FIG. 7 shows the PCR products of A. butzleri, A. butzleri-like, C.upsaliensis, and H. canis.

FIG. 8 shows the hybridization results with each of the species-specificoligo probes used in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention provides a method forpreparing nucleic acid probes for identifying species of bacterialpathogens, and it provides isolated nucleic acid probes which are usefulin identifying and distinguishing between various bacteria includingCampylobacter spp, Arcobacter spp., and Helicobacter spp.

As used herein, the term “isolated” refers to a nucleic acidsubstantially free of cellular material or culture medium when producedby recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. The term “nucleic acid” isintended to include DNA and RNA and can be either double stranded orsingle stranded.

In particular, the inventors have developed species specificoligonucleotide probes using the partial sequence of a specificconserved and essential gene, glyA which encodes serinehydroxymethyltransferase which is referred to herein as SHMT. Toidentify and differentiate closely-related species, a combinedPCR-hybridization strategy was explored using these probes to targetdifferent regions within the glyA gene.

Degenerate oligodeoxyribonucleotides (oligos), designed by comparing theglyA gene sequences of Campylobacter jejuni (Chan, V. L. and Bingham,H., 1990) and Escherichia coli (Sambrook et al., 1989), were used in thepolymerase chain reaction (PCR) to amplify a glyA fragment ofapproximately 640 base pairs (bps) from C. jejuni ATCC 33560, C. coliATCC 33559, C. lari ATCC 35221, C. upsaliensis ATCC 43954, Helicobactercinaedi ATCC 35683, H. pylori (clinical isolate), and Arcobacternitrofigilis ATCC 33309. Alignment of the DNA sequences of these glyAfragments revealed three regions which were used to developspecies-specific oligo probes. Two sets of probes targeting two regionsof glyA were designed to detect and differentiate C. jejuni, C. coli, C.lari, and C. upsaliensis, which are designated CJATC-1, CC-1, CL-1, CU-1for set 1, respectively, and CJATC-2, CC-2, CL-2, CU-2 for set 2,respectively. Another set of probes, targeting the third region, wasdesigned to detect and differentiate H. cinaedi, H. pylori, and A.nitrofigilis. which are designated HC-1, HP-1 and AN-1, respectively. Afurther set of probes were designed to detect and differentiate A.butzleri (GlyA-AB); A. butzleri-like (GlyA-BL and GlyA-BL2);Campylobacter upsaliensis (GlyA-CU) and Helicobacter canis (GlyA-HC andGlyA-HC2). Using the hybridization and washing conditions describedbelow, these probes were found to be species-specific. The probes of thepresent invention have the following nucleic acid sequences:

CJATC-1: 5′-TTTTC CGCAC ACTCA TGTAG TAAGC TCAAC TA-3′ (SEQ ID NO: 1);

CJATC-2: 5′-GAAAA AGTAA GAGAA ATTGC TAAAA AAGAA-3′ (SEQ ID NO: 2);

CC-1: 5′-ATTTC CTCAT GCTCA TGTAG TAAGC TCTAC AA-3′ (SEQ ID NO: 3);

CC-2: 5′-GAAAA AGTTA GGGAA ATTGC TCATA TTGTA-3′ (SEQ ID NO: 4);

CL-1: 5′-ATTCC CTTAT GCTCA TGTTG TAAGT TCT-3′ (SEQ ID NO: 5);

CL-2: 5′-GATAA AGTTA GAGAG ATAGC AAAAG AGATT-3′ (SEQ ID NO: 6);

CU-1: 5′-TTTCC CTCAC GCACA CATCG TAAGC TCA-3′ (SEQ ID NO: 7);

CU-2: 5′-GAAAA AGTAA GAGAA ATAGC ACACA TCGTT-3′ (SEQ ID NO: 8);

HC-1: 5′-TGAGC GCGTG AAGCA GCTAT TTGGC TGTGC GT-3′ (SEQ ID NO: 9);

HP-1: 5′-AGAAA GGGCT AAAAA GCTTT TCAAT TGCCA GT-3′ (SEQ ID NO: 10);

AN-1: 5′-AGATA GAGCT TGTGA AATTT TTGGT TGTAA AT-3′ (SEQ ID NO: 11);

GlyA-AB: 5′-GCT TCT GCA TAC GCA AGA GAA ATT GAT TCA AA- 3′ (SEQ ID NO:12);

GlyA-BL: 5′-GCA AGT GCA TAT GCA AGA GAG ATT GAT TTT AA-3′ (SEQ ID NO:13);

GlyA-BL2: 5′-AAG TAA ACC AAG CTT TTC AGG GCA AAA CTA CTC T-3′ (SEQ IDNO: 14);

GlyA-CU: 5′-GGT TAG TAG CTC GGG TAA AAT GTA TGA AAG C-3′ (SEQ ID NO:15);

GlyA-HC: 5′-CAG GAT TGA TTA CGA CAA GCT ACG CCA AAG CGC GC-3′ (SEQ IDNO: 16); and

GlyA-HC2: 5′-TTC TGC CTA TAC AAG AGA GCT AGA TTT TGC CAA G-3′ (SEQ IDNO: 17).

It will be appreciated that the invention includes probes that arecomplementary to the above probes. The invention also includes nucleicacids having substantial homology or identity with the nucleic acidsequences described above. The term “homologous” means probes that havenucleic acid sequences which have slight or inconsequential sequencevariations from these sequences while maintaining their function as aspecies specific probe. The variations may be attributable to localmutations or structural modifications. The invention also includesnucleic acid probes that have been truncated or contain additionalnucleotide sequences over the nucleic acid sequences described above.

The probes of the invention are useful in detecting and identifyingbacteria in a sample including: biological materials, such as feces,blood or other bodily fluids or tissues from humans or animals such asmammals and poultry; in foods such as dairy products most particularlymilk and poultry; and in environmental samples such as water andindustrial wastes. The sample may be treated using techniques known inthe art to render the nucleic acid molecules in the sample available tohybridize with the nucleic acid probes of the present invention. Onesample may be assayed using several probes, either simultaneously orconsecutively, in order to identify the species of the bacteria in thesample.

A nucleic acid probe of the present invention may be labelled with adetectable marker such as a radioactive label which provides for anadequate signal and has sufficient half-life such as ³²P, ³H, ¹⁴C or thelike. Other detectable markers which may be used include antigens thatare recognized by a specific labelled antibody, fluorescent compounds,enzymes, antibodies specific for a labelled antigen, andchemiluminescent compounds. An appropriate label may be selected havingregard to the rate of hybridization and binding of the probe to thenucleotide to be detected and the amount of nucleotide available forhybridization.

The nucleic acid probe may be used in solution (free) or may be bound toa solid support. Solid supports which may be used include polymericsupports such as polystyrene or agarose beads and filters such as nylonor nitrocellulose filters.

Accordingly, the present invention also relates to a method of detectingthe presence of a specific bacteria in a sample by detecting a nucleicacid that hybridizes with a particular probe of the invention comprisingcontacting the sample under hybridization conditions with one or more ofthe nucleic acid probes of the invention and determining the degree ofhybridization between the nucleic acid molecules in the sample and thenucleic acid probe(s).

Hybridization conditions which may be used in the method of theinvention are known in the art and are described for example in SambrookJ, Fritch E F, Maniatis T. In: Molecular Cloning, A LaboratoryManual,1989. (Nolan C, Ed.), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. The hybridization product may be assayed usingtechniques known in the art. The nucleotide probe may be labelled with adetectable marker as described herein and the hybridization product maybe assayed by detecting the detectable marker or the detectable changeproduced by the detectable marker.

According to one embodiment the present invention provides a method fordetecting the presence of a Campylobacter, Helicobacter or Arcobacterspp. bacteria in a sample comprising: (a) contacting the nucleic acidmolecules of the sample, under hybridization conditions, with one ormore nucleic acid probes selected from the group consisting of: CJATC-1SEQ ID NO: 1; CJATC-2 SEQ ID NO: 2; CC-1 SEQ ID NO: 3; CC-2 SEQ ID NO:4; CL-1 SEQ ID NO: 5; CL-2 SEQ ID NO: 6; CU-1 SEQ ID NO: 7; CU-2 SEQ IDNO: 8; HC-1 SEQ ID NO: 9; HP-1 SEQ ID NO: 10; AN-1 SEQ ID NO: 11;GlyA-AB SEQ ID NO 12; GlyA-BL SEQ ID NO 13; GlyA-BL2 SEQ ID NO 14;GlyA-CU SEQ ID NO 15; GlyA-HC SEQ ID NO 16; and GlyA-HC2 SEQ ID NO: 17;or nucleic acid sequences complementary or homologous to thesesequences, and (b) determining if the nucleic acid molecules in thesample sample hybridizes with the nucleic acid probe(s).

According to another embodiment, the present invention provides a methodfor detecting C.jejuni in a sample by detecting a nucleic acid moleculein the sample that hybridizes with the nucleic acid probe CJATC-1 orCJATC-2, the method comprising contacting the sample under hybridizationconditions with one or more of the nucleic acid probes CJATC-1 orCJATC-2, and determining the degree of hybridization between the nucleicacid molecule in the sample and the nucleic acid probe(s).

In another embodiment, the present invention provides a method fordetecting C.coli in a sample by detecting a nucleic acid molecule in thesample that hybridizes with the nucleic acid probe CC-1 or CC-2, themethod comprising contacting the sample under hybridization conditionswith one or more of the nucleic acid probes CC-1 or CC-2, anddetermining the degree of hybridization between the nucleic acidmolecule in the sample and the nucleic acid probe(s).

In a further embodiment, the present invention provides a method fordetecting C.lari in a sample by detecting a nucleic acid molecule in thesample that hybridizes with the nucleic acid probe CL-1 or CL-2, themethod comprising contacting the sample under hybridization conditionswith one or more of the nucleic acid probes CL-1 or CL-2, anddetermining the degree of hybridization between the nucleic acidmolecule in the sample and the nucleic acid probe(s).

In yet another embodiment, the present invention provides a method fordetecting C.upsaliens in a sample by detecting a nucleic acid moleculein the sample that hybridizes with the nucleic acid probe CU-1, CU-2 orGlyA-CU, the method comprising contacting the sample under hybridizationconditions with one or more of the nucleic acid probes CU-1, CU-2 orGlyA-CU, and determining the degree of hybridization between the nucleicacid molecule in the sample and the nucleic acid probe(s).

According to another embodiment, the present invention provides a methodfor detecting H.cinaedi in a sample by detecting a nucleic acid moleculein the sample that hybridizes with the nucleic acid probe HC-1, themethod comprising contacting the sample under hybridization conditionswith the nucleic acid probe HC-1, and determining the degree ofhybridization between the nucleic acid molecule in the sample and thenucleic acid probe.

In another embodiment, the present invention provides a method fordetecting H.pylori in a sample by detecting a nucleic acid molecule inthe sample that hybridizes with the nucleic acid probe HP-1, the methodcomprising contacting the sample under hybridization conditions with thenucleic acid probe HP-1, and determining the degree of hybridizationbetween the nucleic acid molecule in the sample and the nucleic acidprobe.

In another embodiment, the present invention provides a method fordetecting H. canis in a sample by detecting a nucleic acid molecule inthe sample that hybridizes with the nucleic acid probe GlyA-HC orGlyA-HC2, the method comprising contacting the sample underhybridization conditions with the nucleic acid probe GlyA-HC orGlyA-HC2, and determining the degree of hybridization between thenucleic acid molecule in the sample and the nucleic acid probe.

In a further embodiment, the present invention provides a method fordetecting A.nitrofigilis in a sample by detecting a nucleic acidmolecule in the sample that hybridizes with the nucleic acid probe AN-1,the method comprising contacting the sample under hybridizationconditions with the nucleic acid probe AN-1, and determining the degreeof hybridization between the nucleic acid molecule in the sample and thenucleic acid probe.

In a further embodiment, the present invention provides a method fordetecting A. butzleri in a sample by detecting a nucleic acid moleculein the sample that hybridizes with the nucleic acid probe GlyA-AB, themethod comprising contacting the sample under hybridization conditionswith the nucleic acid probe GlyA-AB, and determining the degree ofhybridization between the nucleic acid molecule in the sample and thenucleic acid probe.

In a further embodiment, the present invention provides a method fordetecting A. butzleri-like in a sample by detecting a nucleic acidmolecule in the sample that hybridizes with the nucleic acid probeGlyA-BL or GlyA-BL2, the method comprising contacting the sample underhybridization conditions with the nucleic acid probe GlyA-BL orGlyA-BL2, and determining the degree of hybridization between thenucleic acid molecule in the sample and the nucleic acid probe.

According to another embodiment of the present invention there isprovided a kit for detecting the presence of a Campylobacter,Helicobacter or Arcobacter bacteria in a sample comprising: (a) one ormore nucleic acid probes selected from the group consisting of: CJATC-1SEQ ID NO: 1; CJATC-2 SEQ ID NO: 2; CC-1 SEQ ID NO: 3; CC-2 SEQ ID NO:4; CL-1 SEQ ID NO: 5; CL-2 SEQ ID NO: 6; CU-1 SEQ ID NO: 7; CU-2 SEQ IDNO: 8; HC-1 SEQ ID NO: 9; HP-1 SEQ ID NO: 10, AN-1 SEQ ID NO: 11,GlyA-AB SEQ ID NO 12; GlyA-BL SEQ ID NO 13; GlyA-BL2 SEQ ID NO 14;GlyA-CU SEQ ID NO 15; GlyA-HC SEQ ID NO 16; and GlyA-HC2 SEQ ID NO: 17;or nucleic acid sequences complementary or homologous to thesesequences; (b) reagents required for hybridization of the nucleic acidprobe with the nucleic acid molecules molecules in the sample; and (c)directions for its use.

According to a further embodiment, this kit can be used for identifyingany one of Campylobacter jejuni (C. Jejuni), Campylobacter coli (C.coli), Campylobacter lari (C. lari) and Campylobacter upsaliens (C.upsaliens); Helicobacter cinaedi (H. cinaedi), Helicobacter pylori (H.pylori), Helicobacter canis (H. canis), Arcobacter nitrofigalis (A.nitrofigalis) Arcobacter butzleri (A. butzleri); and Arcobacterbutzleri- like (A. butzleri-like) in a sample, the method comprising themethod just mentioned and the further step of correlating the nucleicacid probe(s) which hybridize with the identity of the bacteria. This isdiscussed further below in the Discussion under the Examples.

By using the methodology described in the present application, oneskilled in the art can readily isolate and identify specific probes fromall of the other species of the Campylobacter, Helicobacter andArcobacter species as well as other genera and other species ofpathogenic bacteria. In particular, the glyA gene of other bacterialspecies or genera can be amplified using the oligonucleotide primers S1and S2 (described herein) in the PCR. Other primers may also be preparedfrom the glyA sequences disclosed in FIGS. 1 and 6. In addition, theglyA gene can be sequenced from other bacterial genera and suitablenucleotide primers can be prepared.

Accordingly, the present invention provides the preparation of a nucleicacid probe that is specific for a particular species of bacteriacomprising: (a) amplifying a glyA fragment from the bacteria using anoligonucleotide primer; (b) determining the nucleic acid sequence of theamplified fragment; (c) comparing the nucleic acid sequence of theamplified fragment with the nucleic acid sequence of glyA from one ormore different bacterial species, and (d) identifying a nucleic acidsequence that is unique to the particular species of bacteria.

The length and bases of the primers for use in the PCR are selected sothat they will hybridize to different strands of the desired sequenceand at relative positions along the sequence such that an extensionproduct synthesized from one primer when it is separated from itstemplate can serve as a template for extension of the other primer intoa nucleic acid of defined length.

Primers which may be used in the invention are oligonucleotides, i.e.,molecules containing two or more deoxyribonucleotides of the nucleicacid molecule of the invention which occur naturally as in a purifiedrestriction endonuclease digest or are produced synthetically usingtechniques known in the art such as for example phosphotriester andphosphodiester methods (See Good et al Nucl. Acid Res 4:2157, 1977) orautomated techniques (See for example, Conolly, B A. Nucleic Acids Res.15:15(7): 3131, 1987). The primers are capable of acting as a point ofinitiation of synthesis when placed under conditions which permit thesynthesis of a primer extension product which is complementary to theDNA sequence of the invention, i.e., in the presence of nucleotidesubstrates, an agent for polymerization such as DNA polymerase and atsuitable temperature and pH. Preferably, the primers are sequences thatdo not form secondary structures by base pairing with other copies ofthe primer or sequences that form a hair pin configuration. The primermay be single or double-stranded. When the primer is double-stranded itmay be treated to separate its strands before using to prepareamplification products. The primer preferably contains between about 7and 25 nucleotides.

The primers may be labelled with detectable markers which allow fordetection of the amplified products. Suitable detectable markers areradioactive markers such as P-32, S-35, I-125, and H-3, luminescentmarkers such as chemiluminescent markers, preferably luminol, andfluorescent markers, preferably dansyl chloride,fluorcein-5-isothiocyanate, and 4-fluor-7-nitrobenz-2-axa-1,3 diazole,enzyme markers such as horseradish peroxidase, alkaline phosphatase,β-galactosidase, acetylcholinesterase, or biotin.

It will be appreciated that the primers may contain non-complementarysequences provided that a sufficient amount of the primer contains asequence which is complementary to a nucleic acid molecule of theinvention or oligonucleotide sequence thereof, which is to be amplified.Restriction site linkers may also be incorporated into the primersallowing for digestion of the amplified products with the appropriaterestriction enzymes facilitating cloning and sequencing of the amplifiedproduct.

PCR refers to a process for amplifying a target nucleic acid sequence asgenerally described in Innis et al, (Academic Press, 1990) in Mullis elal., (U.S. Pat. No. 4,863,195) and Mullis, (U.S. Pat. No. 4,683,202)which are incorporated herein by reference. Conditions for amplifying anucleic acid template are described in M. A. Innis and D. H. Gelfand,(PCR Protocols, A Guide to Methods and Applications M. A. Innis, D. H.Gelfand, J. J. Sninsky and T. J. White eds, pp3-12, Academic Press1989), which is also incorporated herein by reference.

The process described by Mullis amplifies any desired specificnucleotide sequence contained in a nucleic acid or mixture thereof. Theprocess involves treating separate complementary strands of thenucleotide sequence to be amplified with two oligonucleotide primerswhich are extended under suitable conditions to form complementaryprimer extension products which act as templates for synthesizing thenucleotide sequence. The primers are selected so that they aresufficiently complementary to different strands of each specificnucleotide sequence to be amplified. The steps of the PCR reaction maybe carried out sequentially or simultaneously and the steps may berepeated until the desired level of amplification is obtained.

The amplified products can be isolated and distinguished based on theirrespective sizes using techniques known in the art. For example, afteramplification, the DNA sample can be separated on an agarose gel andvisualized, after staining with ethidium bromide, under ultra violet(uv) light. DNA may be amplified to a desired level and a furtherextension reaction may be performed to incorporate nucleotidederivatives having detectable markers such as radioactive labelled orbiotin labelled nucleoside triphosphates. The primers may also belabelled with detectable markers. The detectable markers may be analyzedby restriction and electrophoretic separation or other techniques knownin the art.

The conditions which may be employed in the methods of the inventionusing PCR are those which permit hybridization and amplificationreactions to proceed in the presence of DNA in a sample and appropriatecomplementary hybridization primers. Conditions suitable for thepolymerase chain reaction are generally known in the art. For example,see M. A. Innis and D. H. Gelfand, PCR Protocols, A guide to Methods andApplications M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. Whiteeds, pp3-12, Academic Press 1989, which is incorporated herein byreference. Preferably, the PCR utilizes polymerase obtained from thethermophilic bacterium Thermus aquatics (Taq polymerase, GeneAmp Kit,Perkin Elmer Cetus) or other thermostable polymerase may be used toamplify DNA template strands.

It will be appreciated that other techniques such as the Ligase ChainReaction (LCR) and NASBA may be used to amplify a nucleic acid moleculeof the invention. In LCR, two primers which hybridize adjacent to eachother on the target strand are ligated in the presence of the targetstrand to produce a complementary strand (Barney in “PCR Methods andApplications”, August 1991, Vol.1(1), page 5, and European PublishedApplication No. 0320308, published Jun. 14, 1989). NASBA is a continuousamplification method using two primers, one incorporating a promotersequence recognized by an RNA polymerase and the second derived from thecomplementary sequence of the target sequence to the first primer (U.S.Ser. No. 5,130,238 to Malek).

The present invention also includes peptides encoded for by the nucleicacid probes of the present invention. Also included in the invention areantibodies that are specific for the peptides of the invention. Suchantibodies may be useful in determining the identity of a bacterialpathogen in a sample. “Antibodies” used herein are understood to includepolyclonal antibodies, monoclonal antibodies, antibody fragments (e.g.,Fab′ and F(ab′)2 ) and recombinantly produced partners. Conventionalmethods can be used to prepare the antibodies. Monoclonal antibodies maybe readily generated using conventional techniques (see U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporatedherein by reference; see also “Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses”, Plenum Press, Kennett, McKearn, andBechtol (eds.), Cold Spring Harbor Laboratory Press, 1988, and Goding,J. W., Monoclonal Antibodies: Principles and Practice, 2nd Ed., AcademicPress, London, 1986 which are also incorporated herein by reference).Due to the small nature of the peptides they will generally be coupledto a carrier to increase their immunogenicity prior to immunization.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1

Bacterial strains, plasmids, and growth conditions used:

Bacterial strains used in this study are listed in Table 1.Campylobacter spp., Helicobacter spp., and Arcobacter nitrofigilis(Table 1) were grown on Columbia Agar Base (Oxoid) supplemented withdefibrinated horse blood (5% final concentration). Campylobacter spp.and Helicobacter spp. were incubated at 37° C. from 24 to 48 hours and36 to 72 hours, respectively, while A. nitrofigilis was incubated atroom temperature (approximately 25° C.) for 24 to 48 hours. All specieswere grown in a 3 L anaerobic jar under microaerophilic conditionscreated by the Campylobacter Gas Generating Kit (Oxoid) which generatesan atmosphere containing approximately 6% oxygen and 10% carbon dioxide.

For gene cloning experiments, plasmid pBluescript II KS+ (Stratagene)and E. coli strain JM101 (Sanger et al., 1977) were used. The E. colicells were grown in Luria Bertani (LB) broth at 37° C. Competent cellswere prepared by the rubidium chloride/calcium chloride protocol andtransformed by standard procedures (Sanger et al., 1977). Transformantswere grown on LB agar supplemented with ampicillin (100 ug/ml finalconcentration).

Extraction of genomic DNA:

Genomic DNA from Campylobacter spp. with the strain designations LMG,RG, and BVA (Table 1) was from P. Vandamme (Gent, Belgium). Genomic DNAfrom the remaining bacterial species was extracted as previouslydescribed (Thompson et al., 1988). Briefly, cultures grown on agarplates were scraped off and washed three times in 1XSSC (150 mM sodiumchloride, 15 mM trisodium citrate, pH 7.0). For each wash, cells werecentrifuged at 5000 rpm (GSA Sorvall rotor) for 5 minutes. Followingcentrifugation, the supernatant was decanted and the cell pellet wasresuspended in 1XSSC. Finally, the washed cells were resuspended in1XSSC containing 27% sucrose to a concentration of approximately 10⁹cells per ml. Proteinase K was then added to a final concentration of0.2 mg/ml and incubated at 60° C. for 1 hour. Genomic DNA was purifiedand extracted with an equal volume of buffer-saturated phenol (50 mMTris.Cl, 10 mM EDTA, pH 8.0) with constant slow agitation for 30 mins atroom temperature. The mixture was then chilled to 0° C. and centrifugedat 5000 rpm for 5 minutes. The phenol (top) phase was removed and theextraction was repeated. The aqueous DNA solution was dialyzed in 1.5 Lof T₁₀E₁ (10 mM Tris.Cl, 1 mM EDTA, pH 8.0), three times, at 4° C. for12 hours. The DNA was then precipitated with 0.3M sodium acetate pH 5.2and two volumes of 95% ethanol for 12 hours at −20° C. The DNA wascentrifuged for 30 minutes at 7000 rpm. The DNA pellet was redissolvedin T₁₀E₁ and stored at 4° C.

Polymerase Chain Reaction (PCR):

A 640 bp region of the glyA gene was chosen to be amplified. It isdirectly flanked by conserved domains identified by amino acid sequencealignment of the C. jejuni, E. coli and available partial sequence ofthe rabbit SHMT homologue (Chan, V. L. and Bingham, H., 1990). Thisregion also encompasses the domain implicated for binding the coenzyme,pyridoxal-5′-phosphate (Sambrook et al., 1989) and a domain that hasbeen suggested to be part of the enzyme's active site (Innis et al.(eds.), 1990; Sambrook et al., 1989). The sequences of the two conservedflanking domains were used to synthesize degenerate oligo primers, S1(5′-AA(C/T) AAA TA(C/A) GC(A/T) GAA GG(T/A) TAT- 3′) and S2 (5′-ATG CAT(C/T)AA (A/T)GG (A/T)CC (A/T)CC TTG- 3′), to amplify the region of theglyA gene of the selected species. The PCR was performed on all theCampylobacter spp., the Helicobacter spp., A. nitrofigilis, B.adolescentis, E. coli, L. casei, P. aeruginosa, and S. sonnei (Table 1)using a thermal cycler (Perkin Elmer Cetus).

The PCR reactions were optimized at a concentration of 1 mM MgCl₂ forall species except for A. nitrofigilis, which was optimized at aconcentration of 2 mM MgCl₂. The components of each 100 ul PCR reactionwere 1 ug of genomic DNA (except for H. pylori for which 0.5 ug was used[as estimated from ethidium bromide stained agarose gels]), 20 pmoles ofeach primer, 20 umoles of deoxyribonucleotide triphosphates, 1Xamplification buffer (10 mM Tris.Cl, 50 mM KCl, pH 8.3), and 2.5 unitsof Taq DNA polymerase (Promega and Boehringer Mannheim). The reactionsolutions were overlaid with 100 ul of mineral oil to prevent anyevaporation. The samples were subjected to 30 cycles of amplification,each of which consisted of template denaturation at 95° C. for 1.5minutes, primer annealing at 42° C. for 2 minutes, and chain extensionat 72° C. for 1 minute. After the 30 cycles, an additional extensionstep at 72° C. for 5 minutes was performed at the end of the reaction.The PCR products were purified from the deoxyribonucleotidetriphosphates by passing the reaction solution through a Sephadex G-50spun column (equilibrated in STE [10 mM Tris.Cl pH 7.5, 10 mM NaCl, 1 mMEDTA]). From each 100 ul PCR, 10 ul was subjected to electrophoresis inan ethidium bromide-stained 1% agarose gel, visualized under uv lightillumination, and photographed.

To test the sensitivity of the PCR/hybridization method, serialdilutions of the C. jejuni ATCC 33560 genomic DNA template ranging from1 fg to 1 ug were used in the PCR reactions.

Cloning, miniprep, and sequencing:

The PCR products of the Campylobacter spp., Helicobacter spp., and A.nitrofigilis (Table 1) were subcloned into pBluescript II KS+ at theEcoRV site and subsequently used to transform E. coli strain JM101competent cells. Plasmid preparations (minipreps) were obtained andpurified for sequencing using the alkaline lysis method (Sanger et al.,1977). The clones were sequenced by the Sanger dideoxy-chain terminationmethod (Schirch et al., 1985) using the Sequenase Version 2.0 DNAsequencing kit (United States Biochemical) according to themanufacturer's recommendations. [alpha-³⁵S]-dATP (1000 Ci/mmole, ICNBiomedicals Canada Ltd.), and the M13(−20) forward and reverse primers(Stratagene) of pBluescript II KS+, and the PCR primers, S1 and S2, wereused for the reactions.

DNA sequence alignment, probe designs, and syntheses:

The nucleotide sequences were analyzed using the Microgenie SequenceAnalysis Program Version 5 (Beckman Instruments, Inc.) and Clustal VMultiple Alignment Program (Higgins et al., 1992; Higgins, D. G. andSharp, P. M., 1989). Alignment of the partial glyA nucleotide sequencesof C. jejuni ATCC 33560, C. coli ATCC 33559, C. lari ATCC 35221, C.upsaliensis ATCC 43954, H. pylori (clinical isolate), H. cinaedi ATCC35683, and A. nitrofigilis ATCC 33309, identified 28 bp and 32 bpregions which were used to design the species-specific oligo probesCJATC-1, CC-1, CL-1, CU-1, HC-1, HP-1 and AN-1 (series 1 probes)(synthesized by Dalton Chemical Laboratories Incorporated), and CJATC-2,CC-2, CL-2 and CU-2 (series 2 probes) (synthesized by ACGT Corporated).

End-labelling of the probes:

The species-specific oligos were radioactively labeled in 20 ulreactions containing 20 pmoles of the oligos, 20 pmoles of[gamma-³²P]-ATP (4500 Ci/mmole, ICN Biomedicals Canada Ltd.), 1XPolynucleotide Kinase buffer (70 mM Tris.Cl pH 7.6, 10 mM MgCl₂, 5 mMdithiothreitol), and using 20 units of T4 Polynucleotide Kinase(Pharmacia and New England Biolabs). The reactions were incubated at 37°C. for 30 minutes and stopped by heating at 65° C. for 15 minutes. Theradioactively-labeled probes were purified by passing the reactionsolution through a STE equilibrated Sephadex G-50 spun column.

Southern blot:

HindIII-digested lambda phage DNA, 100 bp ladder DNA (Pharmacia), andpBluescript II KS+ vector (molecular weight markers and negativecontrols), glyA recombinant plasmid clones (positive control), and thePCR products from all the species examined were electrophoresed in a 1%agarose gel and transferred onto GeneScreen Plus nylon-based membranes(Du Pont Canada Inc.) by vacuum transfer using the LKB 2016 VacuGeneVacuum Blotting System (Pharmacia LKB Biotechnology). The transferprocedure consists of 15 minutes of depurination (2N HCl), 20 minutes ofdenaturation (1.5M NaCl, 0.5M NaOH), 20 minutes of neutralization (1.0MTris.Cl, 2.0M NaCl, pH 5.0), and 1 hour of transfer (20XSSC - 3M NaCl,0.3M trisodium citrate, pH 7.0) under a constant vacuum pressure of 55cm.H₂O.

Southern hybridizations:

After Southern blotting, the membranes were air-dried at roomtemperature for 12 hours. Prior to hybridization, they were soaked in2XSSC and prehybridized at 42° C. and 45° C. (for series 1 and 2 probes,respectively) for 30 minutes in 10 ml of prehybridization solution (1%SDS, 1M NaCl, 10% dextran sulfate, and 5 mg/ml denatured sheared salmonsperm DNA. Then, the labeled probe was added with a specific activity of3×10⁵ cpm/ml and the hybridization was done at 42° C. and 45° C. (forseries 1 and 2 probes, respectively) for 8 to 24 hours. This wasfollowed by two washes, each with 0.2XSSC at 60° C. and 50° C. (forseries 1 and 2 probes, respectively) for 10 minutes with constantagitation. Bands were visualized by autoradiography using X-ray films(X-OMAT AR, Kodak Scientific Imaging Film) exposed to the membranes for40 minutes to 10 hours at room temperature and also, 10 to 20 hours at−70° C.

RESULTS

DNA sequences and alignment—species-specific oligo probes. The completesequences were obtained by merging the sequences from both ends of thesubcloned glyA fragment. Two independent glyA recombinant clones of eachspecies were sequenced to ensure the accuracy of the sequences.

Three regions were chosen to design species-specific rather thangenus-specific probes. The first set of oligo probes to detect C.jejuni, C. coli, C. lari, and C. upsaliensis were designed from theregion suggested to be part of the active site of SHMT (Innis et al.,1990; Sanger et al., 1977), while the oligo probes to detect H. cinaedi,H. pylori, and A. nitrofigilis were designed from a region with highsequence variation located downstream of the conserved domain targetedby the degenerate S1 oligo. The second set of oligo probes to detect thefour Campylobacter spp. were designed from another region of variablesequences which is adjacent to the conserved domain implicated forbinding to the co-enzyme,pyridoxal-5′-phosphate (Sanger et al., 1977).The sequences of the species-specific single-stranded oligo probes(FIG. 1) are:

CJATC-1: 5′-TTTTC CGCAC ACTCA TGTAG TAAGC TCAAC TA-3′ (SEQ ID NO: 1);

CJATC-2: 5′-GAAAA AGTAA GAGAA ATTGC TAAAA AAGAA-3′ (SEQ ID NO: 2);

CC-1: 5′-ATITC CTCAT GCTCA TGTAG TAAGC TCTAC AA-3′ (SEQ ID NO: 3);

CC-2: 5′-GAAAA AGTTA GGGAA ATTGC TCATA TTGTA-3′ (SEQ ID NO: 4);

CL-1: 5′-ATTCC CTTAT GCTCA TGTTG TAAGT TCT-3′ (SEQ ID NO: 5);

CL-2: 5′-GATAA AGTTA GAGAG ATAGC AAAAG AGATT-3′ (SEQ ID NO: 6);

CU-1: 5′-TTTCC CTCAC GCACA CATCG TAAGC TCA-3′ (SEQ ID NO: 7);

CU-2: 5′-GAAAA AGTAA GAGAA ATAGC ACACA TCGTT-3′ (SEQ ID NO: 8);

HC-1: 5′-TGAGC GCGTG AAGCA GCTAT TTGGC TGTGC GT-3′ (SEQ ID NO: 9);

HP-1: 5′-AGAAA GGGCT AAAAA GCTTT TCAAT TGCCA GT-3′ (SEQ ID NO: 10);

AN-1: 5′-AGATA GAGCT TGTGA AATTT TTGGT TGTAA AT-3′ (SEQ ID NO: 11).

The series 1 set of probes has a T_(m) range from 60.5° C. (for AN-1) to72.1° C. (for HC-1) while the series 2 set of probes has a T_(m) rangefrom 54.9° C. (for CJATC-2) to 59.3° C. (for CU-2). The conditions forhybridization and washing were optimized to select for species-specifichybridizations. Thus, two stringent conditions were used based on themelting temperatures of the two series of probes.

Each probe's species-specificity was tested against the bacteria listedin Table 1. This was done by PCR amplifying the glyA fragments usinggenomic DNA from all the species including all the Campylobacter spp.,Helicobacter spp., A. nitrofigilis, B. adolescentis, E. coli, L. casei,P. aeruginosa, and S. sonnei (FIG. 5). However, no PCR products wereobtained from B. adolescentis and L. casei (data not shown). Inaddition, pBluescript II KS+ and the recombinant plasmids that weresequenced, were used in the hybridizations as negative and positivecontrols, respectively.

The results of the hybridizations are shown in FIG. 3, panels (A) to (G)for the series 1 set of probes and in FIG. 4 panels (A) to (D) for theseries 2 set of probes. The CC-1, CU-1, HC-1, HP-1, and AN-1 probes arespecies-specific under the hybridization and washing conditions sinceexposure times between 40 minutes to 20 hours did not show cross-specieshybridization. While the CJATC-1 and CL-1 probes appear to bespecies-specific after exposure times between 40 minutes to 4 hours,there is some cross-hybridization that can be detected after 18 to 20hours of exposure. The CJATC-1 probe cross-hybridized to the PCRproducts of C. coli and the CL-1 probe cross-hybridized to the PCRproducts of A. nitrofigilis. The CC-2 probe is species-specific underthese hybridization and washing conditions since cross-hybridization tothe other species' PCR products is not seen after exposure times of upto 22 hours. The CJATC-2, CL-2, and CU-2 probes also arespecies-specific after 4 hours of exposure. However, there is somecross-hybridization that can be observed after exposure of 22 hours. TheCJATC-2 and CL-2 probes cross-hybridized to the PCR products of some C.upsaliensis strains, while the CU-2 probe cross-hybridized to the PCRproducts of some C. jejuni strains.

Detection of different strains and serotypes. The ability of the C.jejuni, C. coli, C. lari, and C. upsaliensis probes to hybridize todifferent strains and serotypes of the species was also tested. FIGS. 3and 4 (see the “(A)” panels in each) show that CJATC-1 and CJATC-2probes are able to detect 12 other strains and serotypes of C jejuni;the “(B)” panels in FIGS. 3 and 4 show that CC-1 and CC-2 probes areable to detect 9 other C. coli strains; FIGS. 3 and 4 (see the “(C)”panels) show that CL-1 and CL-2 probes are able to detect 13 otherstrains; and the “(D)” panels in FIGS. 3 and 4 show that CU-1 and CU-2probes are able to detect 13 other C. upsaliensis strains.

Sensitivity. The PCR using the S1 and S2 oligos was performed onserially diluted C. jejuni ATCC 33560 genomic DNA to determine theamplification yield and ultimately, the sensitivity of thisPCR/hybridization approach. The CJATC-1 probe was tested for its abilityto detect the lowest amount of the PCR product. The results in FIG. 5show that the lowest amount of genomic DNA required in order to yieldenough PCR product to be detected by the CJATC-1 probe is 4 picograms(4×10⁻¹² grams).

DISCUSSION

Species-specific oligos were designed from the aligned glyA sequencesand their specificity was tested by subsequent hybridizations. PCRproducts were isolated from all the Campylobacter, Helicobacter, andArcobacter spp. and from E. coli, P. aeruginosa, and S. sonnei.

From the hybridization results, the CC-1, CU-1, HC-1, HP-1, and AN-1probes are species-specific under these hybridization and washingconditions since exposure times of up to 20 hrs did not detect anycross-hybridization with any of the other species. However, the CJATC-1probe cross-hybridized to the PCR products of C. coli, and the CL-1probe cross-hybridized to the PCR products of A. nitrofigilis whenexposed for greater than 18 hours.

The second set of probes were tested using different hybridization andwashing conditions. The results demonstrate that the CC-2 probe isspecies-specific since exposure of up to 22 hours did not reveal anycross-hybridization to any of the other species. However, both theCJATC-2 and CL-2 probes cross-hybridized to the PCR products of some C.upsaliensis strains, and the CU-2 probe cross-hybridized to the PCRproducts of some C. jejuni strains with longer exposure times (e.g. 22hours).

Since an unknown sample may contain any one of the species, the use ofdiagnostic set 1 or 2 (Table 2) would resolve the discrepancies due tocross-hybridization. For example, using diagnostic set 1, if an unknownsample was detected by both CJATC-1 and CC-1, since CJATC-1cross-hybridizes weakly to C. coli and CC-1 only detects C. coli, thesample would be determined as C. coli. However, if the unknown samplewould be detected by CJATC-1 but not by CC-1, then by the samededuction, the sample is determined as C. jejuni. Thus,cross-hybridizations would not be a factor for misidentification.Furthermore, cross-hybridization would not affect the identification ofeither C. jejuni or C. lari strains since the C. jejuni probes do notcross-hybridize to the C. lari glyA PCR fragments and vice versa.

The CJATC-1, CJATC-2, CC-1, CC-2, CL-1, CL-2, CU-1, and CU-2 probescould also detect different strains of their various respective species.However, the strength of the hybridizations were varied. This may be dueto minor nucleotide sequence variations between the different strains,which were observed when the glyA sequences of C. jejuni ATCC 43431(Chan, V. L. and Bingham, H., 1990) and C. jejuni ATCC 33560 werealigned (data not shown). From the sequence alignment analysis, C.jejuni ATCC 43431 and C. jejuni ATCC 33560 vary by 2 nucleotides at theCJATC-1 target sequence. None of the probes hybridized to the otherbacterial species such as C. sputorum subsp. bubulus, E. coli, P.aeruginosa, and S. sonnei.

The sensitivity of this PCR/oligo hybridization strategy was determinedby using the CJATC-1 probe targetting C. jejuni ATCC 33560 as the testspecies. The lowest amount of genomic DNA required to yield sufficientPCR product which could be detected by CJATC-1 was 4 picograms (4×10⁻¹²grams). Since the C. jejuni chromosome is approximately 1.8×10⁶ bps,4×10⁻¹² grams corresponds to approximately 2062 copies of template.However, the result that was detected was of 10 ul of the total 100 ulreaction volume. Therefore, the PCR would be able to amplify detectableamounts of product from approximately 200 copies of template.

While isotopic detection systems may have disadvantages (e.g. isotopicdecay, radiation exposure, etc.), this PCR/hybridization strategy can beused as a rapid diagnostic method for detecting the various species ofCampylobacter, Helicobacter, and Arcobacter. Five hours could be used asthe minimum exposure time for species-specific identification. Thisexposure time would not reveal the cross-hybridizing bands. However, aspreviously mentioned, the probes detect different strains of the samespecies with varying signal intensities. Therefore, a further exposure,such as ten hours, could also be done to detect different strainswithout the appearance of cross-hybridizations. In addition,simultaneous use of a combination of the CC-1, CC-2, CU-1, HC-1, HP-1,and AN-1 probes with the CJATC-1, CJATC-2, CL-1, CL-2, and CU-2 probes(e.g. diagnostic set 1 or 2, ref. Table 2) would significantly reducethe likelihood of misidentifications due to cross-hybridizations. Withthe current conditions and limited exposure times, however, all theprobes that have been designed are species-specific and could identifyand differentiate the Campylobacter, Helicobacter, and Arcobacter spp.that were studied.

Example 2

Bacterial strains and its growth conditions

Bacterial strains used in this study are listed in Table 3. 10 strainseach of Arcobacter butzleri, Arcobacter butzleri-like, Campylobacterupsaliensis, and 3 strains of Helicobacter canis were from LCDC.Campylobacter jejuni, Campylobacter coli, Acrobacter nitrofigilis,Helicobacter cinaedi, Shigella sonnei, Escherichia coli, and Pseudomonasaeruginosa were from the American Type Culture Collection (ATCC),Rockville, USA. Campylobacter lari was from Dr. J. L. Penner, Universityof Toronto, Toronto, Ontario, Canada. Helicobacter pylori is a clinicalisolate from Mount Sinai Hospital, Toronto, Ontario, Canada. TheArcobacter, Campylobacter, and Helicobacter were grown on Mueller Hintonagar supplemented with 10% sheep's defibrinated blood, incubated at 37°C. from 2 to 6 days in a 2.5 L anaerobic jar under microaerophilicconditions created by the Campylobacter Gas Generating Kit (Oxoid).

Genomic DNA Extraction

Bacterial cells were collected and genomic DNA isolated with the DNAzolReagent (Gibco BRL). Cells from a densely grown plate were lysed for 10minutes at room temperature with 1 ml of DNAzol reagent, followed bycentrifugation at 13,000 rpm for 10 minutes at 4° C. DNA in thesupernatant was precipitated by the addition of 0.5 ml of 100% ethanoland placed on ice for 20 minutes. DNA was pelleted at 13,000 rpm for 20minutes at 4° C. The DNA precipitate was washed twice with 95% ethanol,dried under vacuum and resuspended in 200 ul 8 mM NaOH for 48 hours. ThepH was adjusted to 7.5 with the addition of 18 ul of 1M HEPES (freeacid). DNA was quantified by optical density readings at 260 nm and 280nm.

Polymerase Chain Reaction

Three degenerate oligo primers, S1 [5′-AA(C/T) AAA TA(C/A) GC(A/T) GAAGG(T/A) TAT- 3′], S2 [5′-ATG CAT (C/T)AA (A/T)GG (A/T)CC (A/T)CC TTG 3′]and S5 [5′-C(G/T)G C(G/A)A T(G/A)T G (G/A)G CAA TAT C(A/T)G C- 3′], weredesigned based on sequences on the conserved regions of glyA so that a640 bp PCR product could be amplified with S1 and S2 and a 460 bpproduct with S1 and S5. The reaction was optimized at 1.5 mM of MgCl₂for all samples. A 50 ul PCR reaction contained 0.4 ug of genomic DNA,50 pmoles of each primers, 10 umoles each of the fourdeoxyribonucleotide triphosphates, 1X amplification buffer (20 mMTris-HCl, 50 mM KCl, pH 8.4), and 2.5 units of Taq DNA polymerase(Boehringer Mannheim). The samples were overlay with 50 ul of lightmineral oil and amplified for 30 cycles in a thermal cycler (PerkinElmer Cetus) with an initial denaturation at 95° C. for 3 minutes. Eachcycle consisted of denaturation at 95° C. for 1.5 minutes, annealing ofprimers at 48° C. for 2 minutes and extension at 72° C. for 1 minute.After the standard 30 cycle PCR amplification reaction, an additionalextension step at 72° C. for 10 minutes was performed. 2.5 ul of theamplified products were ran in a 1.5% agarose gel, stained with ethidiumbromide and visualized on an UV light illuminator.

Sequencing of PCR Products

The amplified products of two strains from each species of Arcobacterbutzleri, Arcobacter butzleri-like, Campylobacter upsaliensis andHelicobacter canis were obtained by the standard PCR reaction using S1and S2 oligo primers. Each PCR product was passed through a MicroSpinS-400 HR column (Pharmacia Biotech). 5 ul of the products were used forsequencing in an Ampli-Cycle Sequencing Kit (Perkin Elmer). Briefly, the30 ul reaction consisted of 5 ul of DNA template, 20 pmoles of oligoprimers, S1, S2 or an internal primer [GlyA-In1 (5′-GAT AAA ATA TTA GGTATG- 3′)], 5 uCi of [a ³²P] dATP, 5 ul of 20 uM dATP/dTTP mix, and 4 ulof 10X cycling mix. 6.5 ul of the mixture is combined with 2 ul of eachof the termination mixes and overlaid with 20 ul of light mineral oil.The sequencing was performed in a 25-cycle reaction, with a denaturationat 95° C. for 1 minute, annealing of primers at 45° C. for 1 minute andextension at 72° C. for 1 minute. At the end of the cycling reaction, 4ul of stop buffer was added and samples were heated at 94° C. for 3minutes prior to loading onto a 6% polyacrylamide sequencing gel.

DNA Sequence Alignment and Probe Design

The nucleotide sequences were analysis with DNAsis (Helix Corporation)and aligned with Clustal W Multiple Alignment program (Higgins et al.,1992; Higgins and Sharp et al. 1989). The partial glyA sequences of twostrains of Arcobacter butzleri, Arcobacter butzleri-like, Campylobacterupsaliensis and Helicobacter canis, were aligned with otherCampylobacter, Arcobacter, and Helicobacter sequences (sequenced byShahnaz Al Rashid from Dr. V. L. Chan's lab). 31-35 mer oligo probeswere designed for each of the four groups of bacteria and tested fortheir specificity.

End-labeling of the Oligo Probes

The oligo probes were radioactively labeled in 30 ul reaction consistingof 50 pmoles of oligo, 25 uCi of [g ³²P] ATP, 1X T4 polynucleotidekinase buffer (70 mM Tris-HCl, 10 mM MgCl₂, 5 mM DTT, pH 7.6 @25° C.),and 20 units of T4 polynucleotide kinase (Pharmacia Biotech). Thereactions were incubated at 37° C. for 30 minutes and stopped by heatingat 65° C. for 10 minutes. The radioactively labeled probes were passedthrough a Sephadex G-25 MicroSpin column (Pharmacia Biotech).

Southern Blot

The PCR products from all the bacteria listed in Table 3, along with 100bp ladder (Pharmacia Biotech) were electrophoresed in a 1.5-% agarosegel and transferred to Hybond membrane (Amersham) by capillary actions.The blot consisted of depurination (0.25N HCl) for 30 minutes,denaturation for 30 minutes (0.4N NaOH and 0.6N NaCl) and neutralizationfor 30 minutes (0.5M Tris-HCl pH 7.5 and 1.5M NaCl). The DNA wastransferred overnight in 10X SSC (1.5M NaCl, 0.15M trisodium citrate, pH7.0).

Southern Hybridization

After blotting, the filters were air-dried at room temperature for 3hours. The filters were prehybridized at 37° C. for 30 minutes with 10ml of prehybridization solution (50% formamide, 1M NaCl, 1% ultra-pureSDS, 10% dextran sulfate). The labeled probe was added to a specificactivity of 1×10⁶ cpm/ml and hybridized overnight at 37° C. for. Thefilters were washed from 55° C. to 64° C. in 0.2X SSC, 1% SDS for 15minutes with constant agitation. DNA hybrid bands were visualized byautoradiography using X-ray film (X-OMAT AR, Kodak Scientific ImagingFilm) with 2 hours to overnight exposure at room temperature.

RESULTS

DNA Sequence Alignment and Species Specific Oligo Probes

The sequences of Arcobacter butzleri, Arcobacter butzleri-like,Campylobacter upsaliensis and Helicobacter canis, were obtained bymerging the sequencing result of S1, S2 and GlyA-In1 primers. Thesequences were compared among each other and the percentages of identityof the nucleotides are shown in Table 4. The other Campylobacter,Arcobacter, and Helicobacter sequences seen in the multiple alignment(FIG. 6) were from Dr. V. L. Chan's lab. The species-specific oligoprobes (Table 5) were designed for Arcobacter butzleri, Arcobacterbutzleri-like, Campylobacter upsaliensis and Helicobacter canis werebased on information from the sequences and the multiple aligrnnent andare as follows:

GlyA-AB: 5′-GCT TCT GCA TAC GCA AGA GAA ATT GAT TCA AA- 3′ (SEQ ID NO:12);

GlyA-BL: 5′-GCA AGT GCA TAT GCA AGA GAG ATT GAT TTT AA-3′ (SEQ ID NO:13);

GlyA-BL2: 5′-AAG TAA ACC AAG CTT TTC AGG GCA AAA CTA CTC T-3′ (SEQ IDNO: 14);

GlyA-CU: 5′-GGT TAG TAG CTC GGG TAA AAT GTA TGA AAG C-3′ (SEQ ID NO:15);

GlyA-HC: 5′-CAG GAT TGA TTA CGA CAA GCT ACG CCA AAG CGC GC-3′ (SEQ IDNO: 16); and

GlyA-HC2: 5′-TTC TGC CTA TAC AAG AGA GCT AGA TTT TGC CAA G-3′ (SEQ IDNO: 17)

Southern Blot and Hybridization

The 33 strains of Arcobacter butzleri, Arcobacter butzleri-like,Campylobacter upsaliensis and Helicobacter canis supplied by LCDC wereamplified with S1 and S5 primers. The remaining species were negativecontrols amplified with S1 and S2 primers (supplied by Shahnaz Al Rashidfrom Dr. V. L. Chan's lab). The gel was blotted and hybridized to thespecies-specific probes. The results of the hybridizations are shown inFIG. 8.

The A.butzleri species-specific probe, GlyA-AB, was able to detect all10 strains of A.butzleri. No cross hybridization was detected with 4hours of exposure time. However, GlyA-AB did hybridize weakly withArcobacter butzleri-like strains with exposure time exceeding 4 hoursand with A.nitrofiglis with exposure time exceeding 20 hours. TheArcobacter butzleri-like probe, GlyA-BL, was able to detect 6A.butzleri-like strains with no cross hybridization detected after 20hours of film exposure. The second Arcobacter butzleri-like probe,GlyA-BL2, was able to strongly hybridize to 6 and weakly hybridize to 2of the Arcobacter butzleri-like strains after 4 hours of film exposure.No cross hybridization was observed after 20 hours of exposure withGlyA-BL2.

The Campylobacter upsaliensis species-specific probe, GlyA-CU,hybridized to all 10 C. upsaliensis strains along with Arcobacterbutzleri strain reference #11556. The hybridization was repeated withtwo other C. upsaliensis oligo probes (previously developed by ShahnazAl Rashid) and Arcobacter butzleri reference #11556 was detected by allthree probes. GlyA-CU was specific and did not have any cross-specieshybridization with 4 hours of exposure time. But overnight exposure didreveal a weak hybridization to C. jejuni, C. coli, C. lari, and H. canisreference #16485. The Helicobacter canis species-specific probes,GlyA-HC and GlyA-HC2, both detected only 2 strains of H. canis. Bothprobes did not cross react with any other species with exposure timeexceeding 24 hours.

Sensitivity of Oligo Probes

The genomic DNA of A. butzleri ATCC 49616, A. butzleri-like reference#13162, C. upsaliensis ATCC 43954, and H. canis ATCC 51401 were seriallydiluted 10-fold in order to determine the sensitivity of the oligoprobes. GlyA-AB was able of detecting one-half of the PCR products of100 pg, corresponding approximately to 46,000 copies of genomic DNA ofA. butzleri ATCC 49616. GlyA-BL and GlyA-BL2 both detected one-half ofthe PCR products of 1 ng or 460,000 copies of genomic DNA from A.butzleri-like reference #13162. Similarly, GlyA-CU was capable ofdetection 1 pg or 460 copies of genomic template. GlyA-HC and GlyA-HC2both were able to detect 1 ng or 460,000 copies of genomic DNA template.

DISCUSSION

The pairwise nucleotide sequence comparison of the PCR GlyA fragments ofthe two strains of A. butzleri and C. upsaliensis indicates a nucleotideidentities exceeding 97%, thus suggesting a high conservation ofnucleotides among different strains of these two species. The nucleotidesequences of the two A. butzleri-like strains (# 13217 and # 13218)showed a nucleotide identity of 94.23%. The nucleotide sequences of thetwo H. canis strains (#16953 and #16485) sequenced show identity below89%. The glyA sequence of A. butzleri and A. butzleri-like shows highhomologues identities in the range of 85.5% to 86.98%. This high degreeof nucleotide identity would contribute to the weak cross hybridizationobserved in the southern blots. The percentage of identity of the GlyAPCR fragment of C. upsaliensis and H. canis with that of other specieswas all below 70%, which contributed to the specificity and enhanced thespecies-specific hybridization of the oligo probes.

The detection of the Arcobacter butzleri strain #11556 by with both theA. butzleri and C. upsaliensis probes suggest that this A. butzleristrain is a variant. A partial sequencing of the GlyA PCR fragmentindicated a 72.02% nucleotide identity with A. butzleri type strain anda 73.21% identity with C. upsaliensis type strain. A. butzleri reference#11556 is the only strain of A. butzleri originated from a water sourcein Thailand. The rest of the A. butzleri strains originated from humanor animals in North America or Europe.

Although the 2 oligo probes for A. butzleri-like are unable to detectall the strains, a combination of the two probes would be able to detect9 out of the 10 strains, FIG. 8. The inability of the probe to hybridizeto all the strains would suggest a high degree of herterogenecity amongthe A. butzleri-like organisms.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements all of which are includedwithin the spirit and scope of the appended claims.

All publications, patents and patent applications herein areincorporated into the present specification by reference in theirentirety to the same extent as if each individual publication, patent orpatent application was specifically and individually indicated to beincorporated by reference in its entirety.

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

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DETAILED FIGURE LEGENDS

FIG. 1. Multiple nucleotide sequence alignment of the partial glyAsequences. Alignment of the sequences from C. jejuni ATCC 33560, C. coliATCC 33559, C. lari ATCC 35221, C. upsaliensis ATCC 43954, H. cinaediATCC 35683, H. pylori (clinical isolate), and A. nitrofigilis ATCC33309. The boxed regions were used to design and synthesize thespecies-specific oligo probes.

FIG. 2. PCR products of all species resolved in a 1% agarose gel andused in the Southern hybridization experiments.

The molecular weight markers are in lanes L (HindIII-digested lambdaphage DNA), and M (100 bp ladder). The PCR products of each bacterialstrain are in the following lanes: lanes 1 to 13 are C. jejuni strainsATCC 33560, ATCC 43429, ATCC 43430, ATCC 43431, ATCC 43432, ATCC 43433,CEPA-3C, COO6-85, INN7383, V48, D594, D603, and D1916; lanes 14, 15 and16 are C. coli ATCC 33559, C. lari ATCC 35221, and C. lari PC 637,respectively; lanes 17 to 26 are C. coli strains ATCC 33559, LMG 7535,LMG 8530, LMG 9853, LMG 9854, LMG 9855, LMG 9856, LMG 9857, LMG 9858,and LMG 9859; lanes 27 to 40 are C. lari strains ATCC 35221, LMG 8845,LMG 8844, LMG 7929, LMG 9887, LMG 9888, LMG 9889, LMG 9913, LMG 9914,LMG 9152, LMG 9253, LMG 11251, 2314 RG, and 2665 BVA; lanes 41 to 54 areC. upsaliensis strains ATCC 43954, 12030, 13064, 13950, 14013, 14080,14506, 14510, 14526, 14529, 14530, 14532, 14967, and 15172; lane 55 to61 are C. sputorum subsp. bubulus ATCC 33562, H. cinaedi ATCC 35683, H.pylori (clinical isolate), A. nitrofigilis ATCC 33309, E. coli ATCC9637, P. aeruginosa ATCC 10145, and S. sonnei ATCC 11803, respectively;and finally, lanes 62 to 68 are the glyA recombinant plasmids of C.jejuni ATCC 33560, C. coli ATCC 33559, C. lari ATCC 35221, C.upsaliensis ATCC 43954, H.cinaedi ATCC 35683, H. pylori (clinicalisolate), and A. nitrofigilis ATCC 33309, respectively. Lanedesignations are maintained for all figures.

FIG. 3. Autoradiographs of the Southern hybridizations testing thespecies-specificity of the series 1 probes. Unless noted, nocross-hybridizations were observed on the autoradiographs taken after 20hours of exposure.

Panel (A). CJATC-1 probe hybridizing to C. jejuni strains.Autoradiograph after 4 hours of exposure. After 21 hours of exposure,cross- hybridization to C. coli was observed.

Panel (B). CC-1 probe hybridizing to C. coli strains. Autoradiographtaken after 2 hours of exposure.

Panel (C). CL-1 probe hybridizing to C. lari strains. Autoradiographtaken after 2 hours of exposure. After 20 hours of exposure,cross-hybridization to A. nitrofigilis was observed.

Panel (D). CU-1 probe hybridizing to C. upsaliensis strains.Autoradiograph taken after 4 hours of exposure.

Panel (E). HC-1 probe hybridizing to H. cinaedi ATCC 35683.Autoradiograph taken after 4 hours of exposure.

Panel (F). HP-1 probe hybridizing to H. pylori (clinical isolate).Autoradiograph taken after 4 hours of exposure.

Panel (G). AN-1 probe hybridizing to A. nitrofigilis ATCC 33309.Autoradiograph taken after 40 minutes of exposure.

FIG. 4. Autoradiographs of the Southern hybridizations testing thespecies-specificity of the series 2 probes. All autoradiographs weretaken after 22 hours of exposure.

Panel (A). CJATC-2 probe hybridizing to C. jejuni strains.Cross-hybridization to C. upsaliensis was observed.

Panel (B). CC-2 probe hybridizing to C. coli strains. Nocross-hybridization was observed.

Panel (C). CL-2 probe hybridizing to C. lari strains.Cross-hybridization to C. upsaliensis was observed.

Panel (D). CU-2 probe hybridizing to C. upsaliensis strains.Cross-hybridization to C. jejuni was observed.

FIG. 5. Sensitivity of the PCR/hybridization strategy.

Panel (A). PCR products resulting from the various amounts (in ug) oftemplate C. jejuni ATCC 33560 genomic DNA used. Lanes a to g are: 1×10⁻⁵ug, 8×10⁻⁶ ug, 6×10⁻⁶ ug, 4×10⁻⁶ ug, 2×10⁻⁶ ug, 1×10⁻⁶ ug, and no DNA.

Panel (B). Autoradiograph of the southern hybridization using theCJATC-1 probe which is detecting 10 ul of the 100 ul PCR using 4×10⁻⁶ ug(4×10⁻¹² grams) template DNA.

FIG. 6: The multiple sequence alignment using ClustalW alignmentprogram. The following abbreviations are used: CU—C. upsaliensis, AB—A.butzleri, BL—A. butzleri-like, and HC—H. canis. The stars below thesequences represent conserved bases. The locations of thespecies-specific oligo probes are boxed.

FIG. 7: The PCR products of A. butzleri, A. butzleri-like, C.upsaliensis, and H. canis using S1-S5 primers and the PCR products ofthe other species using S1-S2 primers are ran on 1.5% agarose gel andblotted onto Hybond membrane. The reference number of each strains areas indicated, along with the 100 bp ladder.

FIG. 8: The hybridization results with each of the species-specificoligo probes. Panel A indicated exposure time of 4 hours and panel Brepresented overnight exposure. The stringency of the washing conditionis 0.2X SSC+0.1% SDS with increasing temperatures. The temperaturesalong with the oligo probe used are indicated on the left. The relevanthybridizing fragments of the corresponding species are also indicatedabove each blot.

TABLE 1 Reference bacteria used in Example 1. Bacteria Strain⁰ BacteriaStrain⁰ Esherichia coli JM101¹ Campylobacter lari ATCC 35221 (type) *⁺ATCC 9637⁺ PC 637⁴⁺ LMG 8845⁶⁺ Campylobacter jejuni ATCC 33560(type)*^(+#) LMG 8844⁶⁺ ATCC 43429* LMG 7929⁶⁺ ATCC 43430* LMG 9887⁶⁺ATCC 43431* LMG 9888⁶⁺ ATCC 43432* LMG 9889⁶⁺ ATCC 43433* LMG 9913⁶⁺CEPA3C²⁺ LMG 9914⁶⁺ COO6-85²⁺ LMG 9152⁶⁺ INN7383²⁺ LMG 9253⁶⁺ V48²⁺ LMG11251⁶⁺ D594³⁺ 2314 RG⁶⁺ D603³⁺ 2665 BVA⁶⁺ D1916³⁺ Campylobacter coliATCC 33559 (type)*+ Campylobacter upsaliensis ATCC 43954 (type)*⁺ LMG7535⁶⁺ 12030 LMG 8530⁶⁺ 13064 LMG 9853⁶⁺ 13950 LMG 9854⁶⁺ 14013 LMG9855⁶⁺ 14080 LMG 9856⁶⁺ 14506 LMG 9857⁶⁺ 14510 LMG 9858⁶⁺ 14526 LMG9859⁶⁺ 14529 LMG 15882⁶⁺ 14530 14532 14967 15172 Campylobacter sputorumATCC 33562 (type)*⁺ subsp. bubulus Helicobacter cinaedi ATCC 35683(type)*⁺ Helicobacter pylori Clinical isolate*⁺ Arcobacter nitrofigilisATCC 33309 (type)*⁺ Bifidobacterium adolescentis ATCC 15703*⁺Lactobacillus casei ⁵⁺ Pseudomonas aeruginosa ATCC 10145* Shigellasonnei ATCC 11803* ⁰ATCC, American Type Culture Collection, Rockville,U.S.A. ¹supE thi Δ(lac-proAB) F′[traD36 proAB⁺ lacI^(q) lacZΔM15](Schirch et al., 1985) ²Clinical isolates ³Hippuricase negative variants(Vandamme et al., 1992) ⁴Obtained from Dr. J. L. Penner, University ofToronto, Toronto, Ontario, Canada ⁵Obtained from Dr. A. Bognar,University of Toronto, Toronto, Ontario, Canada ⁶Obtained from Dr. P.Vandamme, Laboratorium voor Microbiologie, Belgium *strains used for thePCR, subcloned, and sequenced to generate the species-specific oligoprobes ⁺strains used for the PCR and Southern hybridizations todetermine species-specificity of the probes ^(#) C. jejuni type strainused to determine sensitivity of this PCR/hybridization strategy

TABLE 2 Summary of the Southern hybridization results of probespecificity. And the use of seven probes in each diagnostic set 1 or 2to detect and differentiate the different Campylobacter spp.,Helicobacter spp., and A. nitrofigilis. N/A - not applicable. Probes:Detects: Diagnostic Set: CJATC-1 C. jejuni/C. coli 1 CC-1 C. coli 1 CL-1C. lari/A. nitrofigilis 1 AN-1 A. nitrofigilis 1,2 HC-1 H. cinaedi 1,2HP-1 H. pylori 1,2 CU-1 C. upsaliensis 1,2 CJATC-2 C. jejuni/C.upsaliensis 2 CL-2 C. lari/C. upsaliensis 2 CC-2 C. coli 2 CU-2 C.upsaliensis/C. jejuni N/A

TABLE 3 Bacterial strains used in Example 2 Bacteria Strain BacteriaStrain Arcobacter Reference #13217 Arcobacter Reference #13162 butzlerior ATCC 49616 butzleri-like Reference #11556 Reference #13163 Reference#13135 Reference #13128 Reference #13218 Reference #13432 Reference#13443 Reference #13207 Reference #13129 Reference #13209 Reference#13075 Reference #13114 Reference #12052 Reference #13447 Reference#13220 Reference #14064 Reference #11667 Reference #14841 CampylobacterReference #16672 Helicobacter Reference #16953 upsaliensis or ATCC 43954canis or ATCC 51401 Reference #5424 Reference #17656 Reference #14096Reference #16485 Reference #12030 Reference #13950 Reference #13064Reference #12034 Reference #14967 Reference #17501 Reference #17606Campylobacter ATCC 33556 Shigella sonnei ATCC 11803 jejuni CampylobacterATCC 33559 Escherichia coli ATCC 9637 coli Campylobacter PC637Pseudomonas ATCC 10145 lari aeruginosa Arcobacter ATCC 33309nitrofigilis Helicobacter Clinical Isolate pylori Helicobacter ATCC35683 cinaedi

TABLE 4 The percentages of nucleotide identity by pairwise comparison ofpartial glyA sequences AB - Arcobacter butzleri BL - Arcobacterbutzleri-like CU - Campylobacter upsaliensis HC - Helicobacter canis ABAB BL BL CU CU HC HC Strains #13217 #13218 #13432 #13207 #16672 #14096#16953 #16485 AB #13217 100%  98.32%  86.43%  85.50%  68.40%  67.65% 61.33%  61.71% AB #13218 100%  86.98%  86.61%  68.02%  67.10%  61.33% 62.08% BL #13432 100%  94.23%  69.70%  68.58%  61.71%  62.08% BL #13207100%  69.14%  68.48%  61.15%  60.96% CU #16672 100%  97.03%  65.30% 64.56% CU #14096 100%  65.30%  64.56% HC #16953 100%  88.52% HC #16485100%

TABLE 5 The sequences and melting temperature of the species- specificoligo probes NAME SEQUENCE Tm GlyA-AB 5′ -GCT TCT GCA TAC GCA AGA GAAATT GAT TCA AA- 3′ 64.36 GlyA-BL 5′ -GCA AGT GCA TAT GCA AGA GAG ATT GATTTT AA- 3′ 63.27 GlyA-BL2 5′ -AAG TAA ACC AAG CTT TTC AGG GCA AAA CTACTC T- 3′ 65.58 GlyA-CU 5′ -GGT TAG TAG CTC GGG TAA AAT GTA TGA AAG C-3′ 65.52 GlyA-HC 5′ -CAG GAT TGA TTA CGA CAA GCT ACG CCA AAG CGC GC- 3′71.24 GlyA-HC2 5′ -TTC TGC CTA TAC AAG AGA GCT AGA TTT TGC CAA G-3′67.12

17 32 base pairs nucleic acid single linear other nucleic acid unknown 1TTTTCCGCAC ACTCATGTAG TAAGCTCAAC TA 32 30 base pairs nucleic acid singlelinear other nucleic acid unknown 2 GAAAAAGTAA GAGAAATTGC TAAAAAAGAA 3032 base pairs nucleic acid single linear other nucleic acid unknown 3ATTTCCTCAT GCTCATGTAG TAAGCTCTAC AA 32 30 base pairs nucleic acid singlelinear other nucleic acid unknown 4 GAAAAAGTTA GGGAAATTGC TCATATTGTA 3028 base pairs nucleic acid single linear other nucleic acid unknown 5ATTCCCTTAT GCTCATGTTG TAAGTTCT 28 30 base pairs nucleic acid singlelinear other nucleic acid unknown 6 GATAAAGTTA GAGAGATAGC AAAAGAGATT 3028 base pairs nucleic acid single linear other nucleic acid unknown 7TTTCCCTCAC GCACACATCG TAAGCTCA 28 30 base pairs nucleic acid singlelinear other nucleic acid unknown 8 GAAAAAGTAA GAGAAATAGC ACACATCGTT 3032 base pairs nucleic acid single linear other nucleic acid unknown 9TGAGCGCGTG AAGCAGCTAT TTGGCTGTGC GT 32 32 base pairs nucleic acid singlelinear other nucleic acid unknown 10 AGAAAGGGCT AAAAAGCTTT TCAATTGCCA GT32 32 base pairs nucleic acid single linear other nucleic acid unknown11 AGATAGAGCT TGTGAAATTT TTGGTTGTAA AT 32 32 base pairs nucleic acidsingle linear other nucleic acid unknown 12 GCTTCTGCAT ACGCAAGAGAAATTGATTCA AA 32 32 base pairs nucleic acid single linear other nucleicacid unknown 13 GCAAGTGCAT ATGCAAGAGA GATTGATTTT AA 32 34 base pairsnucleic acid single linear other nucleic acid unknown 14 AAGTAAACCAAGCTTTTCAG GGCAAAACTA CTCT 34 31 base pairs nucleic acid single linearother nucleic acid unknown 15 GGTTAGTAGC TCGGGTAAAA TGTATGAAAG C 31 35base pairs nucleic acid single linear other nucleic acid unknown 16CAGGATTGAT TACGACAAGC TACGCCAAAG CGCGC 35 34 base pairs nucleic acidsingle linear other nucleic acid unknown 17 TTCTGCCTAT ACAAGAGAGCTAGATTTTGC CAAG 34

We claim:
 1. An isolated nucleic acid probe comprising at most 34nucleotides and selected from the group consisting of: (a) a nucleicacid probe having a sequence of SEQ ID NO: 1; (b) a nucleic acid probehaving the sequence of SEQ ID NO: 2; (c) a nucleic acid probe having asequence of SEQ ID NO: 3; (d) a nucleic acid probe having a sequence ofSEQ ID NO: 4; (e) a nucleic acid probe having a sequence of SEQ ID NO:5; (f) a nucleic acid probe having a sequence of SEQ ID NO: 6; (g) anucleic acid probe having a sequence of SEQ ID NO: 7; (h) a nucleic acidprobe having a sequence of SEQ ID NO: 8; (i) a nucleic acid probe havinga sequence of SEQ ID NO: 9; (j) a nucleic acid probe having a sequenceof SEQ ID NO: 10; (k) a nucleic acid probe having a sequence of SEQ IDNO: 11; (l) a nucleic acid probe having a sequence of SEQ ID NO: 12; (m)a nucleic acid probe having a sequence of SEQ ID NO: 13; (n) a nucleicacid probe having a sequence of SEQ ID NO: 14; (o) a nucleic acid probehaving a sequence of SEQ ID NO: 15; (p) a nucleic acid probe having asequence of SEQ ID NO: 16; (q) a nucleic acid probe having a sequence ofSEQ ID NO: 17; and (r) a nucleic acid sequence that is fullycomplementary to (a) or (b) or (c) or (d) or (e) or (f) or (g) or (h) or(i) or (j) or (k) or (l) or (m) or (n) or (p) or (q).
 2. An isolatednucleic acid probe for detecting or identifying C. jejuni which consistsof SEQ ID NO: 1, SEQ ID NO: 2, or a nucleic acid sequence fullycomplementary thereto.
 3. An isolated nucleic acid probe for detectingor identifying C. coli which consists of SEQ ID NO: 3, SEQ ID NO: 4, ora nucleic acid sequence fully complementary thereto.
 4. An isolatednucleic acid probe for detecting or identifying C. lari which consistsof SEQ ID NO: 5, SEQ ID NO: 6, or a nucleic acid sequence fullycomplementary thereto.
 5. An isolated nucleic acid probe for detectingor identifying C. upsaliens which consists of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 15 or a nucleic acid sequence fully complementary thereto.6. An isolated nucleic acid probe for detecting or identifying H.cinaedi which consists of SEQ ID NO: 9, or a nucleic acid sequence fullycomplementary thereto.
 7. An isolated nucleic acid probe for detectingor identifying H. pylori which consists of SEQ ID NO: 10, or a nucleicacid sequence fully complementary thereto.
 8. An isolated nucleic acidprobe for detecting or identifying A. nitrofigalis which consists of SEQID NO: 11, or a nucleic add sequence fully complementary thereto.
 9. Anisolated nucleic acid probe for detecting or identfying A. butzleriwhich consists of SEQ ID NO: 12, or a nucleic acid sequence fullycomplementary thereto.
 10. An isolated nucleic acid probe for detectingor identifying A. butzleri-like bacterium which consists of SEQ ID NO:13, SEQ ID NO: 14, or a nucleic acid sequence fully complementarythereto.
 11. An isolated nucleic acid probe for detecting or identifyingH. canis which consists of SEQ ID NO: 16, SEQ ID NO: 17, or a nucleicacid sequence fully complementary thereto.
 12. A method for detectingthe presence of a Campylobacter, Helicobacter or Arcobacter spp.bacteria in a sample comprising: (a) contacting the nucleic acidmolecules of the sample, under hybridization conditions, with one ormore of nucleic add probes: CJATC-1 SEQ ID NO: 1; CJATC-2 SEQ ID NO: 2;CC-1 SEQ ID NO: 3; CC-2 SEQ ID NO: 4; CL-1 SEQ ID NO: 5; CL-2 SEQ ID NO:6; CU-1 SEQ ID NO: 7; CU-2 SEQ ID NO: 8; HC-1 SEQ ID NO: 9; HP-1 SEQ IDNO: 10; AN-1 SEQ ID NO: 11; GlyA-AB SEQ ID NO 12; GlyA-BL SEQ ID NO 13;GlyA-BL2 SEQ ID NO 14; GlyA-CU SEQ ID NO 15; GlyA-HC SEQ ID NO 16;GlyA-HC2 SEQ ID NO 17; or nucleic acid sequences fully complementarythereto and (b) determining if the nucleic acid molecules in the samplesample hybridizes with the nucleic acid probe(s) thereby detecting anyCampylobacter, Helicobacter or Arcobacter bacteria in said sample.
 13. Amethod for identifying any one of Campylobacter jejuni (C. Jejuni),Campylobacter coli (C. coli), Campylobacter lari (C. lari) andCampylobacter upsaliens (C. upsaliens); Helicobacter cinaedi (H.cinaedi), Helicobacter pylori (H. pylori), Helicobacter canis(H. canis),Arcobacter nitrofigalis (A. nitrofigalis) Arcobacter butzleri (A.butzleri); and Arcobacter butzlerii-like(A. butzlerii-like) bacterium ina sample, the method comprising the method of claim 12, and the furtherstep of correlating the nucleic acid probe(s) which hybridize with theidentity of the bacteria.
 14. A kit for detecting the presence of aCampylobacter, Helicobacter or Arcobacter bacteria in a samplecomprising: (a) one or more of nucleic acid probes: CJATC-1 SEQ ID NO:1; CJATC-2 SEQ ID NO: 2; CC-1 SEQ ID NO: 3; CC-2 SEQ ID NO: 4; CL-1 SEQID NO: 5; CL-2 SEQ ID NO: 6; CU-1 SEQ ID NO: 7; CU-2 SEQ ID NO: 8; HC-1SEQ ID NO: 9; HP-1 SEQ ID NO:10; AN-1 SEQ ID NO: 11; GlyA-AB SEQ ID NO12; GlyA-BL SEQ ID NO 13; GlyA-BL2 SEQ ID NO 14; GlyA-CU SEQ ID NO 15;GlyA-HC SEQ ID NO 16; GlyA-HC2 SEQ ID NO: 17; or nucleic acid sequencesfully complementary thereto; (b) reagents required for hybridization ofthe nucleic acid probe with the nucleic acid molecules in the sample;and (c) directions for its use.
 15. The kit of claim 14 for identifyingany one of Campylobacter jejuni (C. Jejuni), Campylobacter coli (C.coli), Campylobacter lari (C. lari) and Campylobacter upsaliens (C.upsaliens); Helicobacter cinaedi (H. cinaedi), Helicobacter pylori (H.pylori), Helicobacter canis(H. canis), Arcobacter nitrofigalis (A.nitrofigalis) Arcobacter butzleri (A. butzleri); or Arcobacterbutzlerii-like(A. butzlerii-like).
 16. A method for detecting C.jejuniin a sample by detecting (a) nucleic acid molecule in the samplecomprising: contacting the nucleic acid molecules of the sample, underhybridization conditions, with one or both of the nucleic acid probesCJATC-1 (SEQ ID NO: 1) or CJATC-2 (SEQ ID NO: 2), or a nucleic acidsequence fully complementary thereto, and (b) determining if the nucleicacid molecules in the samples hybridize with the nucleic acid probe(s),thereby detecting any C. jejuni in said sample.
 17. A method fordetecting C.coli in a sample by detecting a nucleic acid molecule in thesample comprising: contacting the nucleic acid molecules in the sample,under hybridization conditions, with one or both of the nucleic acidprobe CC-1 (SEQ ID NO: 3) or CC-2 (SEQ ID NO: 4), or a nucleic acidsequence fully complementary thereto, and (b) determining if the nucleicacid molecules in the samples hybridize with the nucleic acid probe(s),thereby detecting any C.coli in said sample.
 18. A method for detectingC.lari in a sample by detecting a nucleic acid molecule in the samplecomprising: contacting the nucleic acid molecules in the sample, underhybridization conditions, with one or both of the nucleic acid probeCL-1 (SEQ ID NO: 5) or CL-2 (SEQ ID NO: 6), or a nucleic acid sequencefully complementary thereto, and (b) determining if the nucleic acidmolecules in the samples hybridize with the nucleic acid probe(s),thereby detecting any C.lari in said sample.
 19. A method for detectingC.upsaliens in a sample by detecting a nucleic acid molecule in thesample comprising: contacting the nucleic acid molecules in a sample,under hybridization conditions, with one or more of the nucleic acidprobes CU-1 (SEQ ID NO: 7) or CU-2 (SEQ ID NO: 8), or GlyA-CU (SEQ IDNO: 15) or a nucleic acid sequence fully complementary thereto, and (b)determining if the nucleic acid molecules in the samples hybridize withthe nucleic acid probe(s), thereby detecting any C.upsaliens in saidsample.
 20. A method for detecting H.cinaedi in a sample by detecting anucleic acid molecule in the sample comprising: contacting the nucleicacid molecules in a sample, under hybridization conditions, with thenucleic acid probe HC-1 (SEQ ID NO: 9), or a nucleic acid sequence fullycomplementary thereto, and (b) determining if the nucleic acid moleculesin the samples hybridize with the nucleic acid probe(s), therebydetecting any H.cinaedi in said sample.
 21. A method for detectingH.pylori in a sample by detecting a nucleic acid molecule in the samplecomprising: contacting the nucleic acid molecules in a sample, underhybridization conditions, with the nucleic acid probe HP-1 (SEQ ID NO:10), or a nucleic acid sequence fully complementary thereto, and (b)determining if the nucleic acid molecules in the samples hybridize withthe nucleic acid probe(s), thereby detecting any H.pylori in saidsample.
 22. A method for detecting H. canis in a sample by detecting anucleic acid molecule in the sample comprising: contacting the nucleicacid molecules in a sample, under hybridization conditions, with thenucleic acid probe GlyA-HC (SEQ ID NO: 16), or GlyA-HC2 (SEQ ID NO: 17),or a nucleic acid sequence fully complementary thereto, and (b)determining if the nucleic acid molecules in the samples hybridize withthe nucleic acid probe(s), thereby detecting any H. canis in saidsample.
 23. A method for detecting A.nitrofigilis in a sample bydetecting a nucleic acid molecule in the sample comprising: contactingthe nucleic acid molecules in a sample, under hybridization conditions,with the nucleic acid probe AN-1 (SEQ ID NO: 11), or a nucleic acidsequence fully complementary thereto, and (b) determining if the nucleicacid molecules in the samples hybridize with the nucleic acid probe(s),thereby detecting any A.nitrofigilis in said sample.
 24. A method fordetecting A. butzleri in a sample by detecting a nucleic acid moleculein the sample comprising: contacting the nucleic acid molecules in asample, under hybridization conditions, with the nucleic acid probeGlyA-AB (SEQ ID NO: 12), or a nucleic acid sequence fully complementarythereto, and (b) determining if the nucleic acid molecules in thesamples hybridize with the nucleic acid probe(s), thereby detecting anyA. butzleri in said sample.
 25. A method for detecting A. butzleri- likebacterium in a sample by detecting a nucleic acid molecule in the samplecomprising: contacting the nucleic acid molecules in a sample, underhybridization conditions, with the nucleic acid probe GlyA-BL (SEQ IDNO. 13), or GlyA-BL2 (SEQ ID NO: 14), or a nucleic acid sequence fullycomplementary thereto, and (b) determining if the nucleic acid moleculesin the samples hybridize with the nucleic acid probe(s), therebydetecting any said bacterium in said sample.